A rapid UPLC-MS/MS assay for the simultaneous measurement of fluconazole, voriconazole, posaconazole, itraconazole, and hydroxyitraconazole concentrations in serum.

BACKGROUND: Triazole antifungals are essential to the treatment and prophylaxis of fungal infections. Significant pharmacokinetic variability combined with a clinical need for faster turnaround times has increased demand for in-house therapeutic drug monitoring of these drugs, which is best performed using mass spectrometry-based platforms. However, technical and logistical obstacles to implementing these platforms in hospital laboratories have limited their widespread utilization. Here, we present the development and validation of a fast and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to measure fluconazole, voriconazole, posaconazole, itraconazole, and hydroxyitraconazole in human serum suitable for incorporation into a hospital clinical laboratory. METHODS: Serum samples (20 µL) were prepared using protein precipitation in the presence of deuterated internal standards. Chromatographic separation was accomplished using reversed phase UPLC and analysis was performed using positive-mode electrospray ionization and collision-induced dissociation MS. RESULTS: Total analytical run time was 3 min. All analytes demonstrated linearity (r2>0.998) from 0.1 to 10 µg/mL (1-100 µg/mL for fluconazole), acceptable accuracy and precision (%DEV<15% and %CV<15% at all levels tested), suitable stability under relevant storage conditions, and correlated well with reference laboratory results. CONCLUSIONS: A simple and rapid UPLC-MS/MS method for monitoring multiple triazole antifungals was developed with a focus on the needs of hospital laboratories. The assay is suitable for clinical utilization and management of patients on these medications.

Evaluation of the Waters MassTrak LC-MS/MS Assay for Tacrolimus and a Comparison to the Abbott Architect Immunoassay.

BACKGROUND: Tacrolimus (Prograf, Advagraf, and FK-506) is the most commonly prescribed calcineurin inhibitor after kidney and liver transplantation. The use of tacrolimus (in conjunction with other drugs) has successfully contributed to the maintenance of solid organ allografts; however, it also exhibits toxic side effects. Therapeutic drug monitoring of tacrolimus is used as an aid to achieve drug concentrations within a narrow therapeutic window. METHODS: The Waters MassTrak Immunosuppressants assay (LC-MS/MS) for the quantification of tacrolimus in whole blood was evaluated for precision, linearity, lower limit of quantification, matrix effects, and accuracy. A method comparison with the Abbott Architect Tacrolimus immunoassay was also performed. RESULTS: The mean concentration (nanograms per milliliter) and coefficient of variation for low, mid, and high patient pools were 0.6% ± 19.9%, 16.0% ± 5.4%, and 31.2% ± 5.8%, respectively. The MassTrak assay was linear from 0.5 to 30.0 ng/mL. Although the MassTrak and Architect assays correlated well (R = 0.97) for patient samples, the MassTrak assay displayed an average negative bias of 18.5% versus Architect (range of 0.0%-36.7%). Analysis of a certified tacrolimus reference material in human whole blood [European Reference Materials (ERM)-DA110a, LGC Standards] on both platforms failed to completely explain the observed difference for patient samples. CONCLUSIONS: Two widely used assays for therapeutic drug monitoring of tacrolimus are not in agreement with one another. Care should be exercised when interpreting results generated on these 2 assay platforms.

Standardization of LC-MS for therapeutic drug monitoring of tacrolimus.

BACKGROUND: LC-MS is increasingly used for therapeutic drug monitoring of tacrolimus. A recent summary from an international proficiency-testing scheme demonstrated that the mass spectrometry respondents were the largest method group. However, these methods lack standardization, which may explain the relatively poor interlaboratory agreement for such methods. This study aimed to provide one path toward the standardization of tacrolimus quantification by use of LC-MS. METHODS: A 40-member whole blood tacrolimus proficiency panel was circulated to 7 laboratories, 4 in the US and 3 in Europe, offering routine LC-MS-based quantification of tacrolimus. All laboratories used a common LC-MS platform and followed the manufacturer's instructions that accompanied an LC-MS reagent kit intended for tacrolimus quantification in whole blood samples. Four patient pools were prepared that had sufficient volume to allow comparison with a tacrolimus reference measurement procedure. RESULTS: For the 40-member panel, the standardized MassTrak LC-MS assay demonstrated excellent agreement with a validated LC-MS method used by Analytical Services International (y = 1.02x - 0.02; r = 0.99). The CVs for the pooled patient samples ranged from 2.0% to 5.4%. The mean difference from the reference measurement procedure ranged from 0.4% to 4.4%. CONCLUSIONS: Tacrolimus assay standardization, which must include all facets of the analysis, is necessary to compare patient results between laboratories and to interpret consensus guidelines. LC-MS can provide accurate and precise measurement of tacrolimus between laboratories.